Sensors and methods for the detection of the presence of substances or molecules in a sample, using a solid-phase assay system, have been described previously. Typically, the sample is put in contact with the sensor, allowing analyte present in the sample to bind to the analyte-specific ligand layer of the sensor. For analysis, the sample may be removed from the sensor. The sensor surface is then analyzed for the presence of the analyte. Sensors can be defined as including immunosensors, affinity sensors and ligand binding sensors, each of which is characterized as involving specific mass change activity in connection with determining whether or not certain molecules or substances are present. Sensors are typically efficient at binding the substance of interest (analyte) and highly sensitive and specific to the analyte. The sensor may consist of one or several layers of various chemical and physical compositions. The composition depends on the nature of the analyte and the matrix in which the analyte is contained. These layers may include any combination of: a solid supporting substrate; attachment layer or layers to bind the substrate and/or subsequent layers in the sensor; any number of intermediate layers; a ligand layer that binds specifically to the analyte. Detection of the analyte bound to the sensor can be achieved by several means including, but not limited to, electrochemical, chemical, and optical methods. Detection of the analyte may be enhanced by various means including enzyme amplification, and the use of a mass-enhanced analyte-specific secondary ligand.
A solid substrate, or base, of the sensor has inherent physical, chemical, electrical, or optical properties that are suited specifically to the detection method employed in the assay. A ligand layer is typically provided above the substrate and an analyte to be detected or measured is bound to the ligand layer. The solid support may be used for the direct binding of the analyte to its surface and subsequent detection. However, depending on the composition, complexity, and/or stability of the analyte or the sample in which the analyte is contained, and the nature of the interactions of the sample/analyte with the solid substrate, it may be necessary to add one or several intermediate layers to the solid support.
Attachment layers may be used as intermediate layers between the solid substrate and the ligand layer if, for example, the ligand layer does not adhere to the substrate, or is destroyed, denatured, destabilized or otherwise inactivated upon binding to the substrate. The surface of the intermediate layer in contact with the solid substrate must adhere tightly to the substrate throughout the preparation and use of the test piece. The surface of the intermediate layer opposite the solid substrate must either be suitable for strong binding of either the ligand layer or another intermediate layer. In this manner, and using these considerations for the nature of the intermediate layers, multiple layers may be assembled, the topmost of which is suitable for the binding of the ligand layer.
The ligand layer forms a sensing surface that is receptive specifically to the analyte of interest when the analyte is present in a sample to be tested. The analyte is thus immobilized onto the sensing surface of the sensor and can be detected by any of the methods mentioned above.
Although some multi-layered sensors such as those outlined above have been described previously in the prior art, development of such sensors, in accordance with the present invention, seeks to improve and enhance their sensitivity. The goal of one element of the present invention, in order to improve this sensitivity, is to immobilize a ligand layer that retains maximum binding capacity for a specific analyte. This usually involves the use of an intermediate attachment layer or layers as described above. This multi-layer molecular film is designed specifically to accommodate downward interaction with the substrate, and upward, optimized interaction with the ligand layer.
Detachment, or delamination, of these intermediate layers from the supporting substrate is a serious problem that must be solved to successfully build a multi-layer sensing surface. Delamination occurs between the substrate and an intermediate layer if the composition of these two components is not conducive to a strong physical or chemical interaction between the components. The interaction between the substrate and intermediate layer may be weakened during the manufacture, assembly, transport, preparation or use of the sensor.
Once an attachment layer or layers is produced that is stable to delamination from the solid support, the topmost of the intermediate layers is used to immobilize a ligand layer specific to the analyte of interest. It is critical that the topmost attachment layer optimizes its interaction with the ligand layer in order to provide maximum binding capacity for the analyte of interest and prevent denaturation, deactivation, or inactivation of the ligand layer. These provisions for the immobilization of the ligand layer are essential to the enhanced sensitivity of the test.
Known sensing systems, in addition to multi-layered sensors for immobilizing analyte that may be present in a test sample, include instrumentation for detecting the immobilized analyte. One class of instrumentation, including surface acoustic wave spectroscopy, ellipsometry, and quartz microbalances, measures the change in mass of the sensor upon immobilization of an analyte. Generally speaking, when the analyte is present in the test sample, it can be detected based on a change in mass at the surface as compared to the mass when no analyte is present in the sample.
Optical instruments in this class, as described in the prior art, direct a beam of light through a number of instrument components or elements to the sensing surface that has been previously exposed to the sample being tested. Light is reflected from the sensor, and its reflected properties, including intensity and various optical properties may be measured. Any change in mass of the sensor due to analyte binding is represented by a change in the properties of the reflected light. In particular, measuring changes in polarization state of the reflected light has proven to be a highly sensitive measurement of mass change. Briefly, the sensor may be analyzed by an appropriate instrument that detects and/or measures the presence of the analyte using light reflected from the analyte or light that is transmitted through the analyte.
The main problem associated with instruments that employ these optical techniques for detection of surface bound analyte involves the accuracy of detection. Since extremely small changes in mass may be indicative of the presence of the analyte, it is a goal of the present invention that the instrument be highly sensitive to enable it to detect such mass changes. Additionally, because these instruments are expected to be utilized in a variety of environments outside of well-controlled laboratory settings, it is a further goal that the instrument design take into consideration a number of factors such as component size, durability and automation of instrument operation.
Based on the foregoing factors and considerations, it would be advantageous to devise a sensor system that overcomes such drawbacks or deficiencies of the prior art by providing a system that includes an instrument that readily functions and cooperates with a sensing unit for detection and/or measurement of specific mass change activity due to the presence of an analyte of interest. The instrument of such a system would be highly sensitive and accurate in connection with the detection and/or measurement related to mass change, while the sensing unit of such a system would immobilize a ligand layer that retains, for all necessary purposes, the analyte of interest.